The present invention concerns novel diagnostic methods involving determination of eosinophil cationic protein (ECP) and novel antibodies that can be employed in such methods.
Eosinophil granule proteins have been suggested to mediate their cytotoxicity when in close contact with the target cells (Hamman et al., J. Immunol. 144 (1990) 3166-3173; Capron et al., Eosinophils in Asthma. London, New York, Tokyo: Acadmic Press (1989) 49-60; Robert et al., J. Allergy Clin. Immunol !1991) 1105-1115). ECP is a potent toxin for the larvae of Shistosoma Mansoni (McLaren et al., Parasitol. 88 (1984) 491-503) and a potent neurotoxin when injected into brains of guinea pigs or into the cerebrospinal fluid of rabbits (Fredens et al., J. Allergy Clin. Immunol. 70 (1982) 361-366). ECP is a membrane-active agent and may cause membrane damage. It induces ion flow through artificial bilayers by forming pores (Yong et al., Nature 321 (1985) 613-616).
Many investigators have shown (Olsson et al., Blood 44 (1974) 235-246; Olsson et al., Blood 67 (1986) 498-503; and Gleich et al., Pro. Natl. Acad. Sci. U.S.A. 83 (1986) 3146-3150) that purified ECP is separated on SDS-PAGE into different molecular weight forms (varying from 18 to 21 kDa). Peterson et al. (Eur. J. Haematol. 40 (1988) 415-423) showed that purified ECP from healthy donors was separated on SDS-PAGE into at least three molecular weight forms and their difference in charge and molecular weights were deduced to reflect different amounts of carbohydrates bound to the protein. Nothing is known about the biological activities of these various molecular forms of ECP (iso-ECPs).
It has long been known that eosinophilia is associated with a variety of inflammatory disorders including allergic disease. In such diseases eosinophils and their toxic products are seen in tissue specimens from inflammation foci, for instance asthma (Arm et al., Adv. Immunol. 51 (1992) 323-382). The cells and their granule products seem to be major causes of the tissue destruction, for instance shedding of epithelial cells in the airways (Filley et al., Lancet 3 (July, 1982) 11-16; Venge et al., Am. Rev. Resp. Dis. 138 (1988) 54-57; and Bousquet et al., N. Engl. J. Med 323 (1990) 1033-1039). The pathophysiological changes seen in the asthmatic lung leads to hyperactivity. Even a low grade inflammation seems to render the patients susceptible to a variety of challenges including allergen exposure (Bisgaard et al., J. Allergy Clin. Immunol. 85 (1990) 891-895) and exercise (Venge et al., J. Allergy Clin. Immunol. 88 (1991) 699-704). These conditions may be identified by the monitoring of activated eosinophils and their product ECP in various biological fluids.
Inflammatory conditions like acute asthma are treated by anti-inflammatory drugs (Sheffer, J. Allergy Clin. Immunol 88(Suppl) (1991) 425-534). The decline of inflammation, reflected by the level of ECP in blood samples, such as serum and plasma, may be of value in monitoring the effect of therapy.
ECP levels have been found elevated in other clinical conditions connected with activated eosinophils like atopic dermatitis (Kapp et al., J. Am. Acad. Dermatol. 24 (1991) 555-558), certain infections (Paganelli et al., J. Allergy Clin. Immunol. 88 (1991) 416-418), autoimmune conditions in the joints (Hxc3xa4llgren et al., Ann. Rheum. Dis. 43 (1984) 556-562), gut (Hxc3xa4llgren et al., Am. J. Med 86 (1989) 56-64) and in 30 parasitic disease (Venge et al., In. Eosinophil cationic proteins (ECP and EPX) in health and disease. Eds Yoshida et al., Immunobiology of the eosinophil. New York: Elsevier Biomedical (1983) 163-179).
The invention is based on our discovery that isoforms of ECP (iso-ECPs) may create different biological effects, e.g. the cytotoxicity of ECP varies between isoforms. This has revealed to the inventors that improved human diagnostic methods are feasible by measuring iso-ECPs in biological fluids.
Consequently, the objectives of the invention are to provide novel and improved human diagnostic methods based on iso-ECP measurements, and reagents to be employed in these methods.
One aspect of the invention is a diagnostic method comprising the steps:
(a) measuring specifically the level of ECP in a sample derived from an individual to be diagnosed, and then
(b) comparing the ECP level found with a standard ECP level.
The characterizing feature of this aspect of the invention is that the level of at least one iso-ECP is selectively determined and compared with a reference level of the same iso-ECP. This aspect excludes the determination of the total amount (level) of all iso-ECPs.
The reference level may be the level found for apparently healthy individuals or a level obtained at an earlier occasion for the same individual. If the level found deviates from the normal level of healthy individuals, this is an indication that the individual suffers from some abnormal state, for instance states as described above (increased levels). In case the level differs from the level obtained at an earlier occasion for the same individual, the deviation indicates a change in state (recovery from or worsening of the abnormal state). In the latter case iso-ECP measurements will allow monitoring of medical treatment as discussed above. Decreased levels compared to the normal level may indicate overtreatment with ani-inflammatorials and/or eosinophil deficiency.
Preferred iso-ECPs to be measured are cytotoxic, such as the cytotoxic variant defined in the experimental section.
The expression xe2x80x9cat least one iso-ECP is selectively determinedxe2x80x9d is meant to include also the sum of various isoforms of ECP, but not the total amount of ECP.
The measured level may be expressed as an absolute concentration (for instance xcexcg/L or nmol/L) or as a value relative some other sample constituents, for instance relative total amount of ECP, relative a certain iso-ECP combination or relative some other sample constituents.
The sample is derived from the human individual to be diagnosed and contains eosinophils and/or ECP. Due to the systemic presence of eosinophils, potential samples are broncho alveolar lavage fluid, blood (including serum and plasma samples), urine, cerebrospinal fluid, sputum, faeces, tear fluid and nasal fluid. Blood samples, for instance whole blood, serum and plasma samples, were at the priority date the preferred samples.
The measurement of iso-ECPs can in principle be performed by any method that is able to discriminate one or more iso-ECPs from other ECPs and to provide the sufficient sensitivity, precision and specificity etc. However as indicated in the experimental section, it is believed that immune assays are preferred.
An immune assay according to the invention means that the sample suspected of containing a deviating level of iso-ECPs is brought into contact with an anti-ECP antibody in an assay medium under conditions permitting formation of immune complexes incorporating ECPs and the anti-ECP antibody. Complexes containing the iso-ECPs to be determined is then determined by per se known methods to give a quantitative or qualitative measure of their level in the sample. In this type of assays the complex as such may be measured, or it may be measured by aid of a biospecific affinity reactant labelled with an analytically detectable substance (label), said reactant (and its label) being capable of becoming specifically incorporated into the complex. Suitable biospecific affinity reactants that can be labelled and employed are anti-ECP antibodies, with preference for those reacting specifically with one or more of the iso-ECPs to be measured, anti-antibodies directed against the constant regions of the anti-ECP antibody present in the complex formed, Proteins A and G etc. Examples of detectable substances (labels) that may be used are luminescers, chromophors, fluorophors, enzymes, enzyme substrates, cofactors, coenzymes, radioactive isotopes, particles (metallic or non-metallic), biotin (detected by its reaction with avidin/strepavidin) etc. Some labels change their signal when becoming incorporated into the immune complex while others do not. The former type of labels provides homogeneous immune assays in which there is no need to separate the label incorporated into the complex from the label not incorporated. The latter type of labels demands the separation to be carried out, for instance by insolubilizing the complex into which the label is incorporated (heterogeneous assays). In order to obtain an insolubilized complex that contains the label, precipitating agents, such as polyethylne glycol and insolubilized and insolubilizable biospecific affinity reactants binding to the complex may be used. This latter type of reagents must not insolubilize the labelled biospecific affinity reactant as such. The immune assays to be employed may also be of the competitive or non-competitive type. The latter type are often called sandwich assays because they utilises two antibodies that are able to bind simultaneous to the antigen (in our case ECP).
Known principles are applied to select the appropriate immune assay protocol, for instance homogeneous or heterogeneous variants, order and type of additions and incubation steps etc. The main point is that the amount of reactants added must be such that the amount of label incorporated into the complex or not incorporated into the complex will reflect the level of the iso-ECP(s) to be measured in the sample.
Normal assay conditions are aqueous media with or without water-miscible co-solvents, temperatures within 0-40xc2x0 C. and pH-values within 4-10.
The term anti-ECP antibody (including an anti-iso-ECP antibody) means an antibody preparation reacting specifically with the intended iso-ECPs. An anti-ECP antibody to be used in the inventive diagnostic method has no substantial reaction with other components that may be present in the sample or assay medium. By the term xe2x80x9csubstantially no reactionxe2x80x9d is meant that the antibody has no reactions destroying the intended purpose, in this case the result of the immune assay used.
Unless otherwise specified, the antibody concept above, in particular the anti-ECP/anti-iso-ECP antibody concept, also comprises antibody active fragments and derivatives, including Fab, F(ab)2, Fv, single chain antibody etc, and any other biospecific affinity reactant binding specifically to ECP or selected iso-ECP(s).
In a preferred immune assay variant the anti-ECP antibody is specific only for the iso-ECPs to be measured, with further preference for monoclonal antibody preparations.
In case of using an anti-ECP antibody reacting with all iso-ECPs it is preferred to combine it in an sandwich assay with an anti-iso-ECP antibody that is specific for the iso-ECP(s) to be measured. In other case, the measurement of the relevant iso-ECP complexes has to be based on molecular iso-ECP differences that are retained in the complexes formed. Compare the assay variants presented in EP-A-535,162 (Axis).
At the priority date the sandwich format with emphasis for the heterogeneous variants were preferred. Thus, the preferred assay variants makes use of two anti-ECP antibodies, at least one of them being able to discriminate between the iso-ECP(s) to be measured and other iso-ECPs. In the heterogeneous assay variants, one of the anti-ECP antibodies are or will, during assay, become bound to a solid phase (support) insoluble in the assay medium used.
A second aspect of the invention is an anti-iso-ECP antibody binding specifically to an epitope that is unique for the native form of a cytotoxic isoform of ECP. Thus the inventive antibody in its binding to ECPs is able to discriminate at least one cytotoxic iso-ECP from other iso-ECPs and/or to at least partially inhibit the cytotoxic activity of the iso-ECP(s) to which the antibody binds. See the experimental section. At the priority date the preferred antibody preparation was specifically directed against an iso-ECP that is able to exert cytotoxicity against the erythroleukemic K562 cell line.